Well before symptoms of familial amyotrophic lateral sclerosis surface, motor neurons struggle to move cargo along their axons. A paper in the October 11 Nature Communications offers a new model system to study the defect, and suggests a possible fix. Researchers led by Catherine Verfaillie and Ludo Van Den Bosch, VIB-KU Leuven, Belgium, generated motor neurons derived from patient stem cells carrying mutations in the fused in sarcoma, a.k.a. FUS, gene. Among several previously described defects, the researchers spotted sluggish mitochondria and lazy endoplasmic reticulum vesicles in axons, as well as fewer contacts between the two organelles. The deficits were reversed by treating the cells with inhibitors of histone deacetylase 6, which removes acetyl groups from the microtubule tracks on which the organelles travel.
“The very important aspect of this study is that it used human cells, so we have a representation of patient neurons in culture. We’ve had many promising results from animal models in this field which have not translated to humans,” noted Majid Hafezparast at the University of Sussex in Brighton, U.K. Fen-Biao Gao, University of Massachusetts, Worcester, found the results convincing. He commended the use of multiple patient lines with different mutations. “This is a well-performed study,” he wrote. “Importantly, they generated one pair of isogenic lines with one corrected point mutation to confirm their key findings.”
Axonal transport defects appear in mice carrying mutations that cause amyotrophic lateral sclerosis (ALS), even before birth (Williamson and Cleveland, 1999; Kieran et al., 2015). The defects affect mice carrying ALS mutations in different genes, including SOD1, VAPB, TDP-43, and FUS (Baldwin et al., 2016; Chen et al., 2016). Researchers are beginning to shed light on the cause, but data in human cells are scarce (De Vos and Hafezparast, 2017).
First author Wenting Guo and colleagues generated motor neuron lines from induced pluripotent stem cells derived from skin fibroblasts of three ALS patients carrying the R521H FUS mutation and one patient carrying P525L FUS. Cells from two of the latter’s family members, who did not carry the mutation, served as controls. Seventy to 95 percent of the cells expressed several well-known motor neuron markers and sported very long, motor neuron-like axons. The patient cells also developed typical ALS phenotypes. FUS migrated out of the nucleus into neurites, and the cells fired fewer action potentials than neurons derived from the control volunteers.
The researchers then labelled mitochondria and endoplasmic reticulum (ER) with fluorescent dyes to track their movements. Three weeks after motor neurons formed, the transport of both organelles started failing and continued to worsen over time. “It got to a point where nothing was moving anymore—it was a complete traffic jam,” said Van Den Bosch. “Still, the mitochondria look quite normal under the electron microscope,” he noted, suggesting these FUS mutations disrupt transport rather than overall mitochondrial health. The mutant cells also harbored fewer axonal ER vesicles, and they associated less with mitochondria than did ER vesicles in control cells, suggesting a possible disruption of mitochondrial-associated ER-membranes (MAMs). Among other things, MAMs appear to facilitate intracellular trafficking of both organelles (Krols et al., 2016).
To ensure their observations were due to the FUS mutations, and not to some other variant or an artifact of the cell culture procedure, the researchers corrected the R521H mutation using CRISPR-Cas9. As expected, the disruptions disappeared. The researchers were also able to recreate the transport deficits in neurons derived from human embryonic stem cells engineered to overexpress one of the two FUS mutants. Patrik Verstreken, whose group studies HDAC6 in the same department as Van Den Bosch, wrote that “while this work does not provide an explanation of why FUS affects motor neurons, the work does show the defects are at least present in motor neurons derived from patient cells, a discovery in and of itself very exciting.” However, he noted that it remains an open question if FUS deficits are specific to motor neurons since the author did not examine other types of neurons.
Because Van Den Bosch’s group had previously discovered that blocking histone deacetylase 6 (HDAC6), a.k.a. α-tubulin deacetylase, improved axonal traffic in a mouse model of Charcot-Marie-Tooth disease type 2, they wondered if these inhibitors might clear the traffic jam in FUS-ALS motor neurons (d’Ydewalle et al., 2011). Acetylation of α-tubulin, an HDAC6 substrate, promotes the binding of motor proteins to microtubules (Reed et al., 2006). Indeed, treating the cells overnight with 1μM of Tubastatin A or ACY-738, both HDAC6 inhibitors, rescued the axonal transport defect. Antisense oligonucleotides that knocked down HDAC6 expression by 50 percent had similar effects. “The inhibitors completely reversed the traffic jam for both mitochondria and ER,” said Van Den Bosch. They also corrected the drop in ER-mitochondrial association. As expected, both drugs increased the acetylation levels of α-tubulin. Neither of these drugs is currently being tested in clinical trials for ALS, but Acetylon Pharmaceuticals, Inc., is testing HDAC6 inhibitors for a variety of diseases. Co-author Matthew Jarpe works at the company.
How do these deficits in axonal transport relate to FUS’s other known ways of causing trouble in ALS? “I don’t know, to be honest,” said Van Den Bosch. Normally located in the nucleus, the RNA-binding protein accumulates in the cytosol in ALS, and forms RNA-protein granules that may facilitate accumulation of toxic protein aggregates, both potentially caused by a disruption to transport across the nuclear membrane (e.g., Oct 2016 news). In this study, however, the authors did not see FUS aggregates. They speculate that, because aggregates co-localize with stress granule markers, stress might be a necessary factor for aggregates to form and they did not stress the cultures. Van Den Bosch said that in future studies he plans to repeat his experiments in stressed cells. “In a worst-case scenario, what we are seeing is something quite downstream [in the disease process],” he said. “If something is wrong with nucleocytoplasmic transport, or with stress granules, this burdens the cells such that other things may go wrong, including axonal transport.”
He noted that unraveling the cascade will be complicated, especially considering the many potential interactions. For example, FUS has been reported to regulate HDAC6 expression (Kim et al., 2010).
Regardless of underlying mechanisms, HDAC6 inhibitors could be helpful, said Van Den Bosch. “I don’t think we’ll stop or reverse ALS by blocking HDAC6, but it could extend the life of motor neurons,” he said. HDAC6 inhibitors are currently being tested in cancer clinical trials and Van Den Bosch plans to continue his lab’s studies of HDAC6 inhibitors in mice. Besides Charcot-Marie-Tooth, other diseases may benefit from lowering HDAC6 activity, including Huntington’s, Parkinson’s, and Alzheimer’s (Dompierre et al., 2007; Godena et al., 2014; Govindarajan et al., 2013). And because HDAC6 knockout mice seem to fare well, the strategy might have few serious side effects. “It is interesting that HDAC6 is emerging as a broadly applicable tool to promote axonal transport,” noted Verstreken.
Hafezparast agreed that HDAC inhibitors appear to be good targets, but cautioned that HDAC6 appears to play a role in the clearance of aggregated proteins, including mutant SOD1 (Xia et al., 2014; Kawaguchi et al., 2003).
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